Extraction of Jathropa Curcas Seed Fats and Oil and Identification

The experiment had the objectives of extracting fats and oil from plant sample using 3:2 hexane-isopropanol solvent through Soxhlet method and of identifying and characterizing unknown oil sample using different chemical analyses. It was divided into two major parts: (1) Extraction of Jathropa curcas seed fats and oil, and (2) Identification and characterization of unknown oil. After the fats and oil was extracted from seeds of Jathropa curcas, the percent yield was reported as 40-41%. The specific gravity, refractive index, acid value, iodine value, peroxide value, and the saponification value of the unknown oil sample were determined. The computed values were 1.05, 1.5765 ± 5.7735 x 10-5, 0.328 ± 2.31 x 10-3 mg acid / g oil, 10. g I2 / 100 g oil, 22 ± 2 ml / g oil, and 208 ± 8.49 mg KOH / g oil, respectively. Comparing the experimental results with the theoretical values, the unknown oil sample was identified as Cinnamon oil. 1. Introduction

Oils have been extracted throughout the ages and were regularly used in practical things like wines, creams and perfumes. When the distillation process was developed, more concentrated essences can now be made and various kinds of individual oils have been created and are now available. With better distilling technology available today, complex chemical compounds can now be separated and it’s easier to manipulate the characteristics of oils. (Rangahau, 2001) The fats and oil, in the first part of the experiment, was extracted from Jathropa curcas seeds taken from the town of Guimbal. Jathropa curcas or Physic Nut (locally known as Tuba-tuba or Kasla) is processed for the high-quality biodiesel production. Its seeds are also a source of the highly poisonous Toxalbumin curcin.

After the seeds were dried for about two days, the outer covering was peeled off, cut and shredded into smaller pieces, which were then weighed in preparation for the extraction. The process of extraction and solvent used in the experiment was Sohxlet extraction and the solvent used was 3:2 hexane-isopropanol. After the oils were extracted, the excess solvent was evaporated using the rotary evaporator apparatus, the remaining extractions were left to further evaporate in the fume hood and later placed in a dessicator. The percentage of fats and oil extracted was then determined. The second part of the experiment includes the characterization of the unknown oil sample using the following tests: Determination of Specific Gravity, Determination of Refractive Index, Determination of Acid Value, Determination of Iodine Value, Determination of Peroxide Value, and Determination of Saponification Value. These reported values were used to identify the unknown oil sample.

2. Theory
Aromatic plants possess odorous volatile substances which occur as essential oil, gum exudate, balsam and oleoresin in one or more parts, namely, root, wood, bark, stem, foliage, flower and fruit. The characteristic aroma is due to a variety of complex chemical compounds. If plant contains oil, it also contains fat that is also essential. Fats are consisting of a wide group of compounds that are generally soluble in organic solvents and largely insoluble in water. It is mainly triesters of glycerol and fatty acids. Unlike any other oils, Jathropa curcas seed oil has no pleasing smell and less medical uses due to the fact that the plant contains hydrogen cyanide, a poisonous compound. Research has shown the seeds contain around 20% saturated fatty acids and 80% unsaturated fatty acids, and yield 25%–40% oil by weight. In addition, the seeds contain other chemical compounds, such as saccharose, raffinose, stachyose, glucose,fructose, galactose, and protein. The oil is largely made up of oleic and linoleic acids.

Furthermore, the plant also contains curcasin, arachidic, linoleic, myristic, oleic, palmitic, and stearic acids and curcin. By subjecting the unknown oil to different test, its specific gravity, refractive index, acid value, iodine value, peroxide value, and saponification value were determined. Specific gravity is the ratio of the density of a substance to the density (mass of the same unit volume) of a reference substance. The reference substance is always water for liquids or air for gases. Temperature and pressure must be specified for both the sample and the reference. Pressure is always 1 atm (101.325 kPa). Temperatures for both sample and reference vary from environment to enviroment. Refractive index (represented by the symbol n) is numerical index value that is expressed relative to the speed of light in a vacuum. The index of refraction value of a material is a number that indicates the number of times slower that a light wave would be in that material than it is in a vacuum. Acid value is the mass of sodium hydroxide in milligrams that is required to neutralize one gram of chemical substance.

The acid number is a measure of the amount of carboxylic acid groups in the fats and oils of the sample seed. In a typical procedure, a known amount of sample dissolved in organic solvent is titrated with a solution of sodium hydroxide with known concentration and with phenolphthalein as a color indicator. Using this test, we can quantify the amount of acid present in the fats and oil of the sample used. Another test performed was the iodine value test. The iodine value is the mass of iodine in grams that is consumed by 100 grams of a chemical substance. It can be used to determine the amount of unsaturation contained in fatty acids. This unsaturation is in the form of double bonds which react with iodine compounds.

The higher the iodine value of a substance, the more unsaturated fatty acid bonds are present in a fat. Fats and oils of Jathropa curcas seed oil can also be tested using the peroxide value. Peroxide value of an oil or fat is used as a measurement of the extent to which rancidity reactions have occurred during storage. By using the peroxide value test, one can determine the extent to which spoilage has advanced in Jathropa curcas fats and oil and any other fats and oil. Saponification value represents the number of milligrams of potassium hydroxide or sodium hydroxide required to saponify 1g of fat under the conditions specified. It is a measure of the average molecular weight of all the fatty acids present. Saponification value allows the comparison of the average fatty acid chain length. These tests were performed using proper equipment and apparatus with the guide of an expert.

3. Methodology

Jathropa curcas Seeds Processing

The Jathropa curcas seeds undergo various processes for its preparation for extraction. These processes involve: * Cleaning: The seeds had been cleaned from the surface of the fruit. * Drying: After cleaning process, the seeds had been dried under the sun in an open place for about 2 days to have a complete drying process. * Peeling: Knife and blade were devices used in peeling the outer covering of the seed. * Grinding: The seeds were crushed into fine pieces using mortar and pestle to be prepared for the extraction process.

Operation of the Soxhlet Extractor ([2])

15 grams of ground Jathropa curcas seeds was laid in the solvent-soaked filter paper while setting up the soxhlet apparatus. The Jathropa curcas seeds filled filter paper was placed in the paper thimble and flowed by 3:2 hexane-isopropanol solvent from the condenser to the round bottom flask. The water source was turned on as the solvent continues to flow. The steam bath was also turned on and waited for 6 hours to have a complete extraction process. The extracted oil and solvent were removed from the round bottom flask, transferred in a rotavap flask and undergone a rotary evaporation. The remaining amount of oil and solvent in the flask was collected and transferred in a clean, empty and pre-weighed vial. It was stored for a week to escape the volatile petroleum ether and cooled in the dessicator. The stored vial with oil was weighed and the amount of oil extracted using soxhlet extraction was collected and stored.

Determination of Specific Gravity ([6])

The previously calibrated pycnometer was filled with the sample in such a manner to prevent entrapment of air bubbles after removing the cap of the side arm. Insert the stopper, immerse in water bath at temperature (T) and hold for 30 minutes. Carefully wipe off any oil that has come out of the capillary opening. Remove the bottle from the bath, clean and dry it thoroughly. Remove the cap of the side arm and quickly weigh, ensuring that the temperature does not fall below temperature (T).

Determination of Refractive Index ([4])

The sample was transferred into the glass slide of the refractometer. In order to carry out determination of the refractive index of oils, the prism box was open and a few drops of oil were placed on the ground surface on the lower prism. It was then closed and the box flattened again, making sure that the oil did not flow away. The cross wire of the telescopes was focused by rotating the eye piece and adjusting the mirror so as to get good illumination. By means of the lower knob, the prism box was turned slowly backwards and forwards until the field of view became colored fringe. By means of the upper knob, the compensator was rotated until the colored fringe disappeared and the lighted image showed a sharp edge. The prism box was rotated until the sharp edge was in coincidence with the intersection of the cross-wires in the telescope. The index of refraction was then read off on the scale through the eye piece. The third decimal place in the refractive index could be read directly and the fourth was estimated with an accuracy of about ± 0.0002.

Determination of Acid Value ([2])

25 mL of diethyl ether and 25 mL of ethanol was mixed in a 250 ml beaker. The resulting mixture was added to 4 grams of oil in a 25 ml conical flask and a few drops of phenolphthalein indicator were added to the mixture. The mixture was titrated with 0.1 M NaOH to the end point with consistent shaking for which a dark pink color was observed and the volume of 0.1 M NaOH was noted.

Determination of Iodine Value ([5])

25 mL carbon tetrachloride solvent was added to the sample and was swirled to ensure that it is completely dissolved. 25 mL Wij’s solution was dispensed into flask containing sample, stopper flask, and was swirled to mix. The time was set to 1.5 hours of storing flasks in the dark place for duration of reaction. Flasks were removed from dark; 15 ml KI solution was added and mixed. 150 ml of recently boiled and cooled water was added and gradually titrated with 0.1 M standard Na2S2O3 solution with constant and vigorous shaking or mechanical stirring. Titration was continued until yellow color has almost disappeared. 2ml starch indicator solution was added to the flasks and titration was continued until blue color has just disappeared.

Determination of Peroxide Value ([4])

5 grams of oil sample was weighed into 250 ml glass stoppered Erlenmeyer flask. 30 mL CH3COOH-CHCl3 was added and swirled to dissolve. A 0.5 mL saturated KI solution was added to the solution from Mohr pipet, let stand with occasional shaking for one minute, and 30 mL water was added. The solution was slowly titrated with 0.1 M Na2S2O3 with vigorous shaking until yellow is almost gone. 0.5 ml 1% starch solution was added, and continues titration, shaking vigorously to release all I2 from CHCl3 layer, until blue just disappears.

Determination of Saponification Value ([4])

A 2.5-gramof filtered oil was weighed into 250-300 mL Erlenmeyer flask. 50 ml of alcoholic KOH solution was being pipet into flask; pipet was drained at a definite time. Flask was connected with air condenser and the fat was boiled until completely saponified. The sample was cooled and titrated with 0.5 M HCl, using phenolphthalein as indicator.

The experiment was conducted at a long period of time which took almost a month and the methods and/or tests used were very time consuming. The extraction method used was the Soxhlet method. Jathropa curcas was used as the sample since it is proven that its seeds contains great amount of fats and oils. The seeds were peeled and then cut into pieces so that it will be suitable in the soxhlet extraction. Soxhlet is a technique that places a specialised piece of glassware in-between a flask and a condenser. The refluxing solvent repeatedly washes the solid extracting the desired compound into the flask. Several extractions were run using the Soxhlet method since the heavy molecules comprise the solvent hence slow cycle of extraction was noted. The collected fats and oils with some solvent were run into the rotary evaporator to finally extract the intended fats and oils. 3:2 ratio of hexane and isopropanol was used as solvent since fats and oil are believed to consist of polar and non-polar regions; thus, the polar region will be attracted to the isopropanol molecules while the non-polar
region will be attracted to hexane molecules – for a better and complete fats and oil extraction.

Sohxlet extraction using the 3:2 hexane-isopropanol solvent yielded 40.389 % and 41.271 % of Jathropa curcas seed fats and oils. Other published experiments reported that extraction of Jathropa curcas seed oil using other type of extraction solvent yielded 25% – 40%. Thus, the extraction process using the 3:2 hexane-isopropanol solvent, even if it had taken several hours, produced the maximum possible yield for the said seed oil. Nonetheless, the other focus of the experiment was the subjection to the unknown oil sample into different tests; namely, Determination of Specific Gravity, Determination of Refractive Index, Determination of Acid Value, Determination of Iodine Value, Determination of Peroxide Value and Determination of Saponification Value. The mean acid value of the unknown sample was computed to be 0.328 milligram acid per gram of oil with standard deviation of 2.31 x 10-3. The low percentage suggests that there are low levels of free fatty acids, and also low hydrolytic and lipolytic activities in the oils.

The low Iodine value of the sample, average of 10. gram Iodine per one hundred gram of oil, suggests that it is saturated – which means there are only few double bonds of fatty acids with the iodine compound. An approximate peroxide value of 22 mililiters per gram of oil indicates the absence or low levels of antioxidants. The mean saponification value of the unknown sample was calculated to be 208 milligram KOH per gram of oil with standard deviation of 8.49. The value suggests that the possible fatty acid dominating the sample is Lauric acid (Molecular Weight = 200 g/mol) which is in fact 80 – 90 % present in cinnamon oil. Also, this value simply inflicts that the oil can be used in soap making. The mean specific gravity of the unknown sample was calculated to be 1.05 while the mean reading of the refractive index was 1.5765 with standard deviation of 5.7735 x 10-5. These values are supplementary justifications based on related literatures for the identification of the unknown sample which was determined to be cinnamon oil.

5. Conclusions
1. The Sohxlet extraction of Jathropa curcas seed fats and oil yielded maximum percent yields using 3:2 hexane-isopropanol solvent compared to other extraction solvents (verifying yields from related literatures). 2. Due to accumulated facts and figures, the unknown oil sample was identified as cinnamon oil. 3. Cinnamon oil has a low percentage of acid value, suggesting that there are low levels of free fatty acids, and also has low hydrolytic and lipolytic activities in the oils. 4. The low iodine value suggests that cinnamon oil is less unsaturated, which is good for paint formulation, etc. and from its saponification value, it is considered good for soap making.

6. References

[1] Rangahau, M.K. (2001) Essential oils and their production. Crop and Food Research, New Zealand. [2] Kyari, M.Z. (2008) Extraction and characterization of seed oils. Department of Science Laboratory Technology, Ramat Polytechnic, Maiduguri, Nigeria [3] Edwards, H.G.M. (2005) Analytical Raman spectroscopic study of cacao seeds and their chemical extracts. Retrieved Date: January 11, 2011. Retrieved From: http://www.worldcocoafoundation.org/scientific-research/research-library/pdf/Edwards05 (1bzPhysiologySeedExtracts(172KB.pdf [4] AOAC Official Methods of Analysis (2005) Oils and Fats. AOAC International. [5] A.O.A.C. 17th ed., 2000, Official method 920. 159 – Iodine absorption number of oils and fats / I.S.I. Handbook of Food Analysis (Part XIII – 1984 page 76). [6] A.O.A.C. 17th ed., 2000, Official method 920. 212 – Specific gravity (Apparent) of Oils, Pycnometer method / I.S.I. Handbook of Food Analysis (Part XIII – 1984 page 72)